A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa.
S-adenosyl-L-methionine (AdoMet)-dependent O-methyltransferases (OMTs) catalyze the transmethylation of a variety of phenolics in bacteria, plants, and humans. To rapidly characterize phenolic OMT activities, we adapted Gibbs' reagent, the dye originally used for detecting phenols, to develop a convenient assay method for measuring the catalytic properties of enzymatic transmethylation of phenolics. We demonstrated that Gibbs' reagent reacted with phenolics yielding distinct absorptive characters that we used to further develop the assay to monitor the reactivities of phenolic OMTs. To validate the method, we identified two caffeate/5-hydroxyferulate 3/5-O-methyltransferases (COMTs) from the black cottonwood, Populus trichocarpa. Together with a few other plant type I OMTs, we demonstrated that our Gibbs' reagent-mediated colorimetric assay could reliably determine the functions and kinetic parameters of phenolic OMTs. Because Gibbs' reagent reacting with different regioselectively modified phenolics displays different colorimetric properties, the assay method can be used to monitor both substrate specificity and the regioselectivity of phenolic OMTs.
Bhuiya
Mohammad-Wadud
MW
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
Liu
Chang-Jun
CJ
D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000737:chemistry; D003124:Colorimetry / Q000191:economics / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007097:Imines / Q000737:chemistry; D007700:Kinetics; D008780:Methyltransferases / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010636:Phenols / Q000737:chemistry; D032107:Populus / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity
D016227:Benzoquinones; D007097:Imines; D010636:Phenols; D008780:Methyltransferases; C019608:caffeate O-methyltransferase; C010358:N,2,6-trichloro-4-benzoquinone imine